Table 2.

Accuracy of the calibration standard measurements of the IL-1RA ELISA

		Standard sample Nominal IL-1RA concentration [pg/ml]
		S1	S2	S3	S4	S5	S6	S7	Run valid?b
		4000 (ULOQ)	2000	1000	500	250	125 (LLOQ)	62.5
Acceptance criteriona		≤ 25	≤ 20	≤ 20	≤ 20	≤ 20	≤ 25	≤ 25
Run 1	R1	2.8	1.0	2.2	1.9	1.1	1.1	2.7	Yes
	R2	2.8	0.7	3.8	0.8	4.2	5.7	1.0
Run 2	R1	4.2	0.5	1.3	3.5	1.2	2.1	0.2	Yes
	R2	4.2	0.3	3.1	1.4	1.5	4.0	4.7
Run 3	R1	1.2	0.2	3.6	0.1	31.7	15.3	39.9	No
	R2	1.3	1.1	2.2	1.1	18.9	16.6	36.4
Run 3 (S1 – S6)c	R1	1.2	0.0	3.0	0.3	26.2	28.1	n.a.c	No
	R2	1.3	1.0	1.6	0.7	14.4	29.3	n.a.c
Run 4	R1	0.4	1.2	5.1	15.9	0.4	4.5	10.1	Yes
	R2	0.5	0.1	1.8	1.6	1.1	5.7	7.0
Run 5	R1	n.d.d	1.8	0.8	0.8	2.2	0.9	7.0	Yes
	R2	n.d.d	1.8	0.7	1.2	2.0	3.4	2.8
Run 6	R1	3.3	2.5	1.3	5.7	5.7	0.9	18.5	Yes
	R2	3.3	1.4	4.7	2.1	7.7	8.7	17.2

Accuracy is given as % bias of the back-calculated concentration from the nominal concentration. The back-calculated IL-1RA concentrations are given in supplementary Table 2.

^aas defined in the EMA’s and FDA’s guidelines on bioanalytical method validation [51–54]; i.e., ≤ 20% (≤ 25% at the ULOQ and LLOQ). Values missing the acceptance criterion are highlighted in yellow.

^bAccording to the criteria defined in the EMA’s and FDA’s guidelines on bioanalytical method validation [51–54]; i.e., ≥ 6 standard samples run at least in duplicate, with ≥ 75% of replicates per run and ≥ 50% of replicates per standard sample showing acceptable accuracy.

^cSince in Run 3 both replicates of S7 were above the maximum acceptable bias, a new calibration curve was generated without S7.

^dOD value was above the OD limit of the detector (“overflow”). n.a., not applicable; n.d., not determined; R, replicate;S, standard sample.